Project Summary Inflammatory bowel disease (IBD) affects millions worldwide. IBD can be divided into Crohn?s Disease (CD) and Ulcerative Colitis (UC), both of which are characterized by chronic inflammation in the gut, leading to diarrhea, ulceration, bleeding, and in many cases tumors. Currently, mild to moderate IBD is treated orally with aminosalicylates (5-ASA), while more severe cases require the use of systemic immunosuppressants with serious side effects. Anti-TNF biologics are effective for the treatment of moderate to severe IBD, but their high cost and immunosuppressive side effects limit their use, especially in children. The goal of this proposal is to develop a more targeted approach ? a non-absorbable TNF inhibitor that can be delivered orally directly to sites of intestinal inflammation. Specifically, we propose to identify and optimize D-peptide inhibitors of TNF-?. D-peptides, the mirror images of natural L-peptides, cannot be digested by proteases and, therefore, they survive transit through the gut. Furthermore, they have the potential for long in vivo half-lives, and they have low immunogenicity. They can readily disrupt protein interfaces with high potency and specificity compared to small molecules and are much less expensive to produce than antibodies. Navigen and the Kay lab have extensive experience using an enantiomeric screening technique (mirror- image phage display) coupled with structure-based design to develop highly potent and specific D-peptide inhibitors (e.g., viral entry inhibitors against HIV, Ebola, and respiratory syncytial virus). Our anti-HIV D-peptide was the first potent and specific D-peptide inhibitor to be discovered and is in advanced preclinical trials. Since D-peptides are not absorbed from the gut and survive GI tract transit, they are ideal for the proposed oral/topical inhibition of TNF-? in the gut mucosa. Inhibitory D-peptides targeting TNF-? would be ideal, because 1) their resistance to proteolysis will enable oral/topical delivery, 2) they have minimal immunogenicity, 3) they effectively block protein-protein interactions, 4) they have a much lower cost of production (vs. antibodies), and 5) TNF?s trimeric structure will allow us to make trimeric D-peptide inhibitors with extreme affinity and potency (taking advantage of avidity). In this 18 month SBIR grant, we will first identify D-peptides that target our chemically-synthesized D-TNF- ?. This will be followed by structural characterization and optimization to create a potent trimeric D-peptide inhibitor of TNF-?, with which we will demonstrate activity in a mouse model of IBD. Our primary goal is to develop a potent D-peptide TNF-? inhibitor that will advance to more sophisticated animal models and IND- enabling studies in Phase II. In addition to providing a valuable and more cost-effective option for IBD treatment, this work will open the door to other localized applications for an anti-TNF-? D-peptide. Finally, success in this project will greatly enable development of D-peptides for other cellular targets.